The proposed research will investigate the process by which neurotropic herpesviruses deliver their genomes to cells. More specifically a dually labeled virus will be used to determine if two viral proteins, the portal and UL25, play important roles in the attachment of HSV-1 capsids to nuclear pore complexes (NPCs) and DNA uncoating. Two hypotheses will be tested. First the idea that the portal mediates capsid binding to NPCs will be tested. Portals and antibodies specific for the portal will be syringe loaded into cells. The cells will subsequently be infected with HSV-1. HSV-1 capsids and viral DNA will be monitored by fluorescence as they move in the infected cells to determine if, in the presence of these proteins, the capsids are still able to bind to nuclei. If portals play a direct role in contacting host NPCs then purified portals and portal antibodies should block binding. Further, electron microscopy will be used to determine the capsid's orientation at the NPC. This will provide evidence to support or contradict the hypothesis that capsids must orient portals toward the NPC to ensure proper DNA uncoating. Second the role of UL25 in capsid binding and DNA uncoating will be investigated. UL25 and antibodies specific for UL25 will be tested for the ability to inhibit capsid-NPC interactions. This will again be done by syringe loading the proteins into cells, infecting the cells, and monitoring the capsids and viral DNA. If UL25 is directly involved in contacting NPCs one would expect these compounds to inhibit binding. Also, the hypothesis that UL25 is a DNA retention protein will be tested. For this, the state of UL25 will be evaluated with two techniques. Western blotting will be used to determine if UL25 is proteolytically digested when a capsid binds to a nuclear pore complex. Electron microscopy will be used to determine the amount of UL25 remaining capsid associated following DNA uncoating. Gaining a better understanding of the binding process will be important for the creation of safer and more efficient herpes based vectors for gene therapy and could lead to the development of new antiviral therapies for the many human diseases caused by these viruses. Relevance Herpes viruses cause a number of human diseases. Investing how these viruses deliver their DNA to initiate infection could lead to new antiviral therapies. Understanding the process could also improve herpes based gene therapy. [unreadable] [unreadable] [unreadable]